SUMMARY: Force and geometry sensing are the major mechanisms by which cells are able to respond to their mechanical environment and give rise to the final form of the tissue and the organism. Many diseases are associated with malfunction of the mechanical sensing and response functions, including cancer and cardiovascular disease. We have determined that the cytoskeleton can transduce force into biochemical signals in the absence of a plasma membrane through stretch-dependent activation of tyrosine phosphorylation. Since tyrosine kinases and phosphatases are involved in the transformation of cancer cells, they are logical candidates for the transduction process. Our recent studies have shown that stretching of the major tyrosine kinase substrate, p130Cas, activates it for tyrosine phosphorylation both in vitro and in vivo. We now plan to study the mechanisms of mechanotransduction at the single molecule level, using a novel single molecule assay with magnetic beads to apply defined forces to stretch the molecules. These studies will measure the dynamics of stretch- dependent binding and phosphorylation as well as the effects of other p130Cas binding molecules. In related studies, we will examine the force-dependence of talin stretching and vinculin binding. This represents another class of force sensors that rely upon domain unfolding to expose alpha helices that bind to other proteins, in this case to vinculin. Mutational as well as steered molecular dynamics studies will direct these studies to probe important aspects of the interaction. Photoactivated localization microscopy (PALM) can define the position of two different photoactivated-fluorophore-tagged proteins to within 5-10 nm. Using this technology, we can measure the relative positions of two proteins or the N- and C-termini of dually tagged proteins such as p130Cas. The stretching of p130Cas in vivo will be measured as well as the geometry of other protein-p130Cas complexes that signal in vivo during periodic edge contractions and matrix contact maturation. We have lined substrates with 20-50 nm fibronectin lines where the dynamics can be viewed relative to the matrix binding sites. Of particular interest is the question of what is the temporal sequence of stretching, phosphorylation and binding interactions during periodic contractions and matrix contact maturation. We can determine if p130Cas is stretched in vivo, is it processively phosphorylated or dephosphorylate, and do binding proteins such as c-Crk form stable complexes. These studies will provide a quantitative description of the force-dependent response pathways that form the basis of in vivo organ development, regeneration, and certain cancers.